The family of nitric oxide synthases (NOS) catalyzes the conversion of L-arginine to L-citrulline and nitric oxide (NO), an important cellular messenger molecule, which has been implicated in a variety of pathophysiological conditions such as septic shock, inflammatory dysfunction, neurodegenerative disease states, and, most importantly, cardiovascular disease. However, despite the increased understanding of the role of NO in contributing to various conditions, effective therapies still await the complete understanding of structure-function relationships within NOS. This understanding is imperative for the modulation of endogenous NO production in vivo, through the design of more effective regulatory drugs. It is proposed that the NOS active site possesses unique synergetic structural-function and electronic properties (which work together both dynamically and cooperatively) specifically tuned for the NOS enzyme functionality. The complexities of this system require the use of sophisticated computational methodologies, in order to fully understand and characterize the functioning mechanism of this enzyme. However, to date, a full-length comprehensive computational investigation, using first principle quantum mechanical (QM) and composite quantum mechanical and molecular mechanics (QM/MM) methodologies, coupled with molecular dynamics (MD) simulation and a variety of other computational analysis tools, has yet to be utilized. Therefore, the intent of this proposal is three fold: (1) to study the NOS environment through MD simulations, in order to fully characterize the dynamic behavior of the tetrahydrobiopterin (H4B) cofactor (a timely electron source for arginine oxidation), whose radical species has been previously identified in electron spin resonance experiments, (2) to analyze the enzyme environment that facilitates the formation of this radical species through QM and QM/MM calculations and electron transfer pathway modeling, in order to identify the key molecular players within the cavity which promote the electron transfer from the cofactor to the heme ferrous-dioxy intermediate, (3) to investigate and elucidate, through computation, the roles of these key players in the electron transfer within NOS, in attempts to direct future experimental and pharmacological interventions into the inhibition of this important enzyme. Primary focus is placed on how Arg199 modulates the electron transfer in NOS a role only revealed during the dynamics of the system. [unreadable] [unreadable]